Combination immunotherapy for the treatment of cancer

ABSTRACT

Agonists to ICOS in combination with a blocking agent to a T cell inhibitory receptor (e.g., CTLA-4, PD-1, etc.) are demonstrated herein to be useful for the treatment of tumors.

RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.15/145,780, filed on May 3, 2016, which is a divisional of U.S.application Ser. No. 14/250,272, filed on Apr. 10, 2014, now U.S. Pat.No. 9,375,475, which is a divisional of U.S. application Ser. No.13/498,570, filed on Sep. 30, 2010, now U.S. Pat. No. 8,709,417, whichis the U.S. National Stage of International Application No.PCT/US2010/051008, filed on Sep. 30, 2010, published in English, whichclaims the benefit of U.S. Provisional Application No. 61/247,438, filedon Sep. 30, 2009. The entire teachings of U.S. application Ser. Nos.14/250,272 and 15/145,780 are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to methods and compositions forthe treatment of cancer employing T cell inhibitory receptor blockade inconjunction with ICOS stimulation.

BACKGROUND OF THE INVENTION

Optimal T cell activation requires contemporaneous signals through the Tcell receptor and costimulatory molecules. CD28, the prototypicalcostimulatory molecule, upon interaction with its ligands B7-1 and B7-2,plays a crucial role in initial T cell priming. Sharpe et al., Nat. Rev.Immunol. 2:203-209 (2002). CD28-mediated T cell expansion is opposed byanother B7-1,2 counter receptor, cytotoxic T lymphocyte associatedantigen 4 (CTLA-4), which attenuates the proliferation of recentlyactivated T cells. Krummel et al., J. Exp. Med. 183:2533-2540 (1996);Leach et al., Science 271:1734-1736 (1996). Temporal regulation of CD28and CTLA-4 expression maintains a balance between activating andinhibitory signals and ensures the development of an effective immuneresponse, while safeguarding against the development of autoimmunity.Blockade of the inhibitory signals mediated by CTLA-4 has been shown toenhance T cell responses and induce tumor rejection in a number ofanimal models, and monoclonal antibodies to human CTLA-4 have foundmodest success in ongoing human clinical trials, including durablecomplete responses in a small subset of patients with metastaticdisease. See, e.g. Korman et al, Adv. Immunol. 90:297-339 (2006).

The identification and characterization of additional CD28 and B7 familymembers PD-1 (programmed death-1), PD-L1 (programmed death ligand-1 orB7-H1), and PD-L2 (B7-DC) has added further complexity to the process ofT-cell activation and peripheral tolerance in humans. Similar to theB7-1,2/CTLA-4 interaction, PD-1 interactions with PD-L1 and PD-L2downregulate central and peripheral immune responses. Fife et al.,Immunol. Rev. 224:166-82 (2008). Accordingly, antibody-based blockade ofPD-1, like CTLA-4, is also being explored in human clinical trials forthe treatment of cancer. See, e.g., Berger et al. Clin. Cancer Res.14:3044-3051 (2008). Nevertheless, as with CTLA-4, improved therapiesare still needed.

Inducible costimulator (ICOS) is a T-cell-specific surface molecule thatis structurally related to CD28 and CTLA-4. Hutloff et al., Nature397:263-266 (1999); Dong et al., Nature 409:97-101 (2001). Initially,the role of ICOS in immune responses was strongly linked to theproduction of Th2 cytokines, suggesting that ICOS-expressing T cellsmight play a role in suppressing immune responses. ICOS-deficient micedemonstrated decreased production of the Th2 cytokine interleukin 10,and IL-10 production by regulatory T cells has been associated with thesuppression of effector T cell responses in a cell-extrinsic manner.Yoshinaga et al., Nature 402:827-832 (1999); Kohyama et al., Proc. Natl.Acad. Sci. USA 101:4192-97 (2004). Contrarily, however, more recent datasuggested that ICOS-expressing T cells might also be involved inautoimmune responses, and CTLA-4 blockade in bladder cancer patients wasshown to increase ICOS expression on CD4+ T cells, which cells thenproduced IFN-gamma and recognized tumor antigen. Yu et al. Nature450:299-303 (2007); Liakou et al., Proc. Natl. Acad. Sci. USA105:14987-992 (2008). Further, ICOS has also been shown to be associatedwith increased survival of both effector memory and regulatory T cells,demonstrating that its functional relevance may not be restricted toregulatory T cells. Burmeister et al., J. Immunol. 180:774-782 (2008).As such, the physiological role of ICOS signaling in the T cellactivation process is still being unraveled. Due to this continuinguncertainty, the potential impact of modulating ICOS signaling in thecontext of cancer therapy is currently unknown.

SUMMARY OF INVENTION

The present invention clarifies the role of ICOS signaling in theprogression or treatment of cancer by demonstrating that thecontemporaneous administration of an ICOS agonist in conjunction with Tcell inhibitory receptor blockade can further enhance the anti-tumoreffects of the blockade. Accordingly, compositions and methods areprovided combining the blockade of a T cell inhibitory receptor (e.g.,CTLA-4 and/or PD-1) with agonist-induced ICOS signaling for thetreatment of cancer. Function-activating ICOS antibodies are provided aswell as ICOS-Ligand-expressing vaccines for use in the subjectcompositions and methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the agonistic effect of anti-ICOS antibody (7E. 17G orC398.4) on murine CD4⁺ T cells in the absence or presence of anti-CD3antibody.

FIG. 2 shows the inverse correlation in untreated animals or animalstreated with anti-CTLA-4 antibody after three weeks between tumor volume(mm³; y-axis) and percent (%) ICOS expression by CD4⁺Foxp3⁻ cells(x-axis).

FIG. 3 demonstrates the percent survival of B16 tumor bearingICOS⁺/ICOSL⁺ animals that were untreated, ICOS⁺/ICOSL⁺⁻ animals treatedwith GVAX and anti-CTLA-4 antibody (9H10), ICOS⁻/ICOSL⁺ animals thatwere untreated, ICOS⁻/ICOSL⁺ animals that were treated with GVAX andanti-CTLA-4 antibody (9H10), ICOS⁺/ICOSL⁻ animals that were untreated,and ICOS⁺/ICOSL⁻ animals treated with GVAX and anti-CTLA-4 antibody(9H10).

FIG. 4 shows tumor size (mm³; y-axis) 0-50 days after tumor challenge(x-axis) in animals treated with GVAX and anti-CTLA-4 antibody (αCTLA4)or animals treated with GVAX, anti-CTLA-4 antibody (αCTLA4) andanti-ICOS antibody (αICO).

FIG. 5 shows percent survival of B16/BL6 tumor bearing animals that wereuntreated, treated with GVAX, treated with GVAX and anti-CTLA-4antibody, or treated with GVAX, anti-CTLA-4 antibody and anti-ICOSantibody.

FIG. 6 shows percent survival of B16/BL6 tumor bearing animals that wereuntreated, treated with GVAX and anti-ICOS (7E.17G9) antibody, treatedwith GVAX and anti-PD-L1 antibody (10F.9G2), or treated with GVAX,anti-PD-L1 antibody and anti-ICOS antibody.

FIG. 7 shows the individual tumor growth curves of each animal (leftcolumn), average tumor volumes in each treatment group (upper rightcorner), and survival curves of each treatment group (bottom rightcorner) of animals treated with GVAX and B16/BL6 cells transduced toexpress Thy1.1 (B16-Thy1.1) or B16/BL6 cells transduced to expressmembrane-bound ICOSL (B16-mICOSL). The numbers in the individual tumorgrowth curves indicate the percentage of tumor-free mice at the end ofthe experiment. For the survival curves, a mouse was considered deadwhen the tumor volume reached 300 mm³.

FIG. 8 shows the individual tumor growth curves of each animal (leftcolumn), average tumor volumes in each treatment group (upper rightcorner) and survival curves of each treatment group (bottom rightcorner) of animals treated with GVAX and anti-CTLA-4 antibody (9H10)alone or in combination with B16/BL6 cells transduced to expressmembrane-bound ICOSL (mICOSL). The numbers in the individual tumorgrowth curves indicate the percentage of tumor-free mice at the end ofthe experiment. For the survival curves, a mouse was considered deadwhen the tumor volume reached 300 mm³.

FIG. 9 shows individual tumor growth curves from a first experiment ofB16/BL6 in mice that were untreated or treated with B16/BL6 cellstransduced to express Thy1.1 (B16-Thy1.1) in the absence or presence ofanti-CTLA-4 antibody (9H10) or B16/BL6 cells transduced to expressmembrane-bound ICOSL (B16-mICOSL) in the absence or presence ofanti-CTLA-4 antibody (9H10). The numbers indicate the percentage oftumor-free mice at the end of the experiment.

FIG. 10 shows individual tumor growth curves from a second experiment ofB16/BL6 in mice that were untreated or treated with B16/BL6 cellstransduced to express Thy1.1 (B16-Thy1.1) in the absence or presence ofanti-CTLA-4 antibody (9H10) or B16/BL6 cells transduced to expressmembrane-bound ICOSL (B16-mICOSL) in the absence or presence ofanti-CTLA-4 antibody (9H10). The numbers indicate the percentage oftumor-free mice at the end of the experiment.

FIG. 11 shows the survival curves of each treatment group of B16/BL6 inmice that were untreated or treated with B16/BL6 cells transduced toexpress Thy1.1 (B16-Thy1.1) in the absence or presence of anti-CTLA-4antibody (9H10) or B16/BL6 cells transduced to express membrane-boundICOSL (B16-mICOSL) in the absence or presence of anti-CTLA-4 antibody(9H10).

FIG. 12A shows average tumor growth curves of B16/BL6 in mice that wereuntreated or treated with B16/BL6 cells transduced to expressmembrane-bound ICOSL (B16-mICOSL) and/or anti-CTLA-4 antibody (9H10).FIG. 12B shows the survival curves of each treatment group of B16/BL6 inmice that hat were untreated or treated with B16/BL6 cells transduced toexpress membrane-bound ICOSL (B16-mICOSL) and/or anti-CTLA-4 antibody(9H10). For the survival curves, a mouse was considered dead when thetumor volume reached 300 mm³.

DETAILED DESCRIPTION

Described herein is the finding that stimulation of ICOS-mediatedsignaling, e.g., via ICOS ligand or an agonist antibody, enhances theanti-tumor effects of blocking agents to T cell inhibitory receptorssuch as CTLA-4 and PD-1. Accordingly, provided herein are compositionscomprising a blocking agent to a T cell inhibitory receptor and an ICOSstimulating agent, and methods of using such compositions to treat apatient afflicted with cancer.

Blocking Agents to T Cell Inhibitory Receptors/Stimulating Agents toICOS

Inducible T cell co-stimulator (ICOS) is also known as “AILIM,” “CD278,”and “MGC39850”. The complete cDNA sequence of ICOS has the GENBANKaccession number of NM_012092.3 and the amino acid sequence of humanICOS has GENBANK accession number of NP_036224. ICOS belongs to the CD28and CTLA-4 cell-surface receptor family. It forms homodimers and playsan important role in cell-cell signaling, immune responses, andregulation of cell proliferation. However, the role of ICOS signaling inmediating anti-tumor responses is currently unknown.

An ICOS ligand (ICOSL) is also referred to as “B7H2,” “GL50,” “B7-H2,”“B7RP1,” “CD275,” “ICOSLG,” “LICOS,” “B7RP-1,” “ICOS-L”, and “KIAA0653.”The complete cDNA sequence of ICOSL has the GENBANK accession number ofNM_015259.4 and the amino acid sequence of human ICOSL has the GENBANKaccession number of NP_056074.

Stimulating agents to ICOS are molecules that generally bind to theextracellular domain of ICOS (e.g., ICOSL). Usually the binding affinityof the blocking agent will be at least about 100 μM. The stimulatingagent will be substantially unreactive with related molecules to ICOS,such as CD28 and other members of the immunoglobulin superfamily. Asdemonstrated herein, suitable stimulating agents activate signaling ofICOS and result in a corresponding increase in T cell activation (e.g.,proliferation). See, e.g. FIG. 1.

Candidate ICOS stimulating agents are screened for their ability to meetthis criteria. Assays to determine affinity and specificity of bindingare known in the art, including competitive and non-competitive assays.Assays of interest include ELISA, RIA, flow cytometry, etc. Bindingassays may use purified or semi-purified ICOS, or alternatively may useT cells that express ICOS, e.g. cells transfected with an expressionconstruct for ICOS; T cells that have been stimulated throughcross-linking of CD3 and CD28; the addition of irradiated allogeneiccells, etc. As an example of a binding assay, purified ICOS may be boundto an insoluble support, e.g. microtiter plate, magnetic beads, etc. Thecandidate stimulating agent and soluble, labeled ICOS ligand are addedto the cells, and the unbound components are then washed off. Theability of the stimulating agent to compete with the natural ligand forICOS binding may be determined by quantitation of bound, labeled ligand.

A functional assay that detects T cell activation may be used forconfirmation that the agent is a stimulating agent of ICOS. For example,a population of T cells may be stimulated with the candidate stimulatingagent in the presence and absence of anti-CD3, as exemplified herein andin FIG. 1. An agent that stimulates ICOS will cause an increase in the Tcell activation, as measured by, e.g. CD4+ T cell proliferation and/orcell cycle progression, release of IL-2, upregulation of CD25 and CD69,etc. It will be understood by one of skill in the art that expression onthe surface of a cell, packaging in a liposome, adherence to a particleor well, etc. will increase the effective valency of a molecule.

A T cell inhibitory receptor as used herein includes any receptorexpressed on the surface of T cells which, when activated or bound byligand, downregulates activation of the T cell. In other words, blockingthe T cell inhibitory receptor enhances T cell activation and/oreffector T cell responses. T cell inhibitory receptors and their ligandsare well-known in the art. Non-limiting and exemplary T cell inhibitoryreceptors include CTLA-4 and PD-1. An skilled artisan will recognizethat the ligands for CTLA-4 include CD80 and CD86. Further, a skilledartisan will recognize that the ligands for PD-1 include PD-L1 andPD-L2.

The complete cDNA sequence of human CTLA-4 has the GENBANK accessionnumber L15006. The region of amino acids 1-37 is the leader peptide;38-161 is the extracellular V-like domain; 162-187 is the transmembranedomain; and 188-223 is the cytoplasmic domain. Variants of thenucleotide sequence have been reported, including a G to A transition atposition 49, a C to T transition at position 272, and an A to Gtransition at position 439. The complete DNA sequence of mouse CTLA-4has the EMBL accession number X05719 (Brunet et al. (1987) Nature328:267-270). The region of amino acids 1-35 is the leader peptide.

The complete cDNA sequence of human PD-1 has the GENBANK accessionnumber NM_005018 and the amino acid sequence of human PD-1 has GENBANKaccession number NP_005009.1. The region of amino acids 1-20 is thesignal peptide, and the mature peptide is found at amino acids 21-288.

Blocking agents to a T cell inhibitory receptor are generally moleculesthat specifically bind to the extracellular domain the T cell inhibitoryreceptor or the extracellular domain of the T cell inhibitory receptorligand to prevent activation of the T cell inhibitory receptor, e.g., byblocking the binding of the T cell inhibitory receptor to its ligand,e.g. CD80, CD86, PD-L1, PD-L2, etc. Usually the binding affinity of theblocking agent will be at least about 100 μM. The blocking agent will besubstantially unreactive with related molecules to the T cell inhibitoryreceptor, such as CD28 and other members of the immunoglobulinsuperfamily. Further, blocking agents do not activate signaling of the Tcell inhibitory receptor. Conveniently, this is achieved by the use ofmonovalent or bivalent binding molecules. It will be understood by oneof skill in the art that the following discussions of cross-reactivityand competition between different molecules is intended to refer tomolecules having the same species of origin, e.g. human T cellinhibitory receptor binds human T cell inhibitory receptor ligand, etc.

Candidate blocking agents are screened for their ability to meet thiscriteria. Assays to determine affinity and specificity of binding areknown in the art, including competitive and non-competitive assays.Assays of interest include ELISA, RIA, flow cytometry, etc. Bindingassays may use purified or semi-purified T cell inhibitory receptorprotein, or alternatively may use T cells that express the T cellinhibitory receptor, e.g. cells transfected with an expression constructfor the T cell inhibitory receptor; T cells that have been stimulatedthrough cross-linking of CD3 and CD28; the addition of irradiatedallogeneic cells, etc. As an example of a binding assay, purified T cellinhibitory receptor protein is bound to an insoluble support, e.g.microtiter plate, magnetic beads, etc. The candidate blocking agent andsoluble, labeled T cell inhibitory receptor ligand are added to thecells, and the unbound components are then washed off. The ability ofthe blocking agent to compete with the ligand for T cell inhibitoryreceptor binding is determined by quantitation of bound, labeled ligand.

Generally, a soluble monovalent or bivalent binding molecule will notactivate T cell inhibitory receptor signaling. A functional assay thatdetects T cell activation may be used for confirmation. For example, apopulation of T cells may be stimulated with irradiated allogeneic cellsexpressing the T cell inhibitory receptor ligand, in the presence orabsence of the candidate blocking agent. An agent that blocks T cellinhibitory receptor signaling will cause an increase in the T cellactivation, as measured by proliferation and cell cycle progression,release of IL-2, upregulation of CD25 and CD69, etc. It will beunderstood by one of skill in the art that expression on the surface ofa cell, packaging in a liposome, adherence to a particle or well, etc.will increase the effective valency of a molecule.

A blocking agent to a T cell inhibitory receptor or a stimulating agentto ICOS may each individually be a peptide, small organic molecule,peptidomimetic, soluble ligands, antibody, or the like. Antibodies are apreferred blocking agent or stimulating agent. Antibodies may bepolyclonal or monoclonal; intact or truncated, e.g. F(ab′)₂, Fab, Fv;xenogeneic, allogeneic, syngeneic, or modified forms thereof, e.g.humanized, chimeric, etc.

In many cases, the blocking agent to a T cell inhibitory receptor orstimulating agent to ICOS will be an oligopeptide, e.g. antibody orfragment thereof, etc., but other molecules that provide relatively highspecificity and affinity may also be employed. Combinatorial librariesprovide compounds other than oligopeptides that have the necessarybinding characteristics. Generally, the affinity will be at least about10⁻⁶, more usually about 10⁻⁸M, i.e. binding affinities normallyobserved with specific monoclonal antibodies.

A number of screening assays are available for blocking agents to a Tcell inhibitory receptor or stimulating agents to ICOS. The componentsof such assays will typically include the T cell inhibitory receptor(and optionally a T cell inhibitory receptor activating agent, e.g. theT cell inhibitory receptor ligand) or ICOS, respectively. The assaymixture will also comprise a candidate pharmacological agent. Generallya plurality of assay mixtures are run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e. at zero concentration or below the level ofdetection.

Conveniently, in these assays one or more of the molecules will bejoined to a label, where the label can directly or indirectly provide adetectable signal. Various labels include radioisotopes, fluorescers,chemiluminescers, enzymes, specific binding molecules, particles, e.g.magnetic particles, and the like. Specific binding molecules includepairs, such as biotin and streptavidin, digoxin and antidigoxin etc. Forthe specific binding members, the complementary member would normally belabeled with a molecule which provides for detection, in accordance withknown procedures.

One screening assay of interest is directed to agents that eitherinterfere with the activation of a T cell inhibitory receptor by itscognate ligands(s) or that activate ICOS signaling. Quantitation ofactivation may achieved by a number of methods known in the art. Forexample, T cell activation may be determined by quantitating cellproliferation, release of cytokines, etc.

Other assays of interest are directed to agents that block the bindingof the T cell inhibitory receptor to its counter-receptor(s) or ligand.The assay mixture will comprise at least a portion of the naturalcounter-receptor, or an oligopeptide that shares sufficient sequencesimilarity to provide specific binding, and the candidatepharmacological agent. The oligopeptide may be of any length amenable tothe assay conditions and requirements, usually at least about 8 aa inlength, and up to the full-length protein or fusion thereof. The T cellinhibitory receptor may be bound to an insoluble substrate. Thesubstrate may be made in a wide variety of materials and shapes e.g.microtiter plate, microbead, dipstick, resin particle, etc. Thesubstrate is chosen to minimize background and maximize signal to noiseratio. Binding may be quantitated by a variety of methods known in theart. After an incubation period sufficient to allow the binding to reachequilibrium, the insoluble support is washed, and the remaining labelquantitated. Agents that interfere with binding will decrease thedetected label.

Candidate blocking or stimulating agents encompass numerous chemicalclasses, though typically they are organic molecules, preferably smallorganic compounds having a molecular weight of more than 50 and lessthan about 2,500 daltons. Candidate blocking or stimulating agentscomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl, sulfhydryl or carboxyl group, preferablyat least two of the functional chemical groups. The candidate blockingor stimulating agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate blocking orstimulating agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

Candidate blocking or stimulating agents are obtained from a widevariety of sources including libraries of synthetic or naturalcompounds. For example, numerous means are available for random anddirected synthesis of a wide variety of organic compounds andbiomolecules, including expression of randomized oligonucleotides.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or readily produced.Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means. Known pharmacological agents may be subjected todirected or random chemical modifications, such as acylation,alkylation, esterification, amidification to produce structural analogs.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal protein-DNAbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.may be used.

Suitable antibodies for use as blocking agents or stimulating agents maybe obtained by immunizing a host animal with peptides comprising all ora portion of the T cell inhibitory receptor or ICOS protein,respectively. Suitable host animals include mouse, rat sheep, goat,hamster, rabbit, etc. The origin of the protein immunogen may be mouse,human, rat, monkey etc. The host animal will generally be a differentspecies than the immunogen, e.g. mouse T cell inhibitory receptor usedto immunize hamsters, human T cell inhibitory receptor to immunize mice,etc. The human and mouse T cell inhibitory receptor contain highlyconserved stretches in the extracellular domain (Harper et al. (1991) J.Immunol. 147:1037-1044). Peptides derived from such highly conservedregions may be used as immunogens to generate cross-specific antibodies.

The immunogen may comprise the complete protein, or fragments andderivatives thereof. Preferred immunogens comprise all or a part of theextracellular domain of human T cell inhibitory receptor (e.g., aminoacid residues 38-161 of human CTLA-4) or ICOS protein, where theseresidues contain the post-translation modifications, such asglycosylation, found on the native T cell inhibitory receptor.Immunogens comprising the extracellular domain are produced in a varietyof ways known in the art, e.g. expression of cloned genes usingconventional recombinant methods, isolation from T cells, sorted cellpopulations expressing high levels of the immunogen, etc.

Where expression of a recombinant or modified protein is desired forproduction of an immunogen, a vector encoding the desired portion of theT cell inhibitory receptor or ICOS protein will be used. Generally, anexpression vector will be designed so that the extracellular domain ofthe T cell inhibitory receptor or ICOS protein is on the surface of atransfected cell, or alternatively, the extracellular domain is secretedfrom the cell. When the extracellular domain is to be secreted, thecoding sequence for the extracellular domain will be fused, in frame,with sequences that permit secretion, including a signal peptide. Signalpeptides may be exogenous or native. A fusion protein of interest forimmunization joins the extracellular domain of the T cell inhibitoryreceptor to the constant region of an immunoglobulin. For example, afusion protein comprising the extracellular domain of a murine T cellinhibitory receptor or ICOS protein joined to the hinge region of humanCg1 (hinge-CH2-CH3) domain may be used to immunize hamsters.

When the T cell inhibitory receptor or ICOS protein immunogen is to beexpressed on the surface of the cell, the coding sequence for theextracellular domain will be fused, in frame, with sequences encoding apeptide that anchors the extracellular domain into the membrane and asignal sequence. Such anchor sequences include the native T cellinhibitory receptor or ICOS protein transmembrane domain, ortransmembrane domains from other cell surface proteins, e.g. CD4, CD8,sIg, etc. Mouse cells transfected with the human T cell inhibitoryreceptor gene or the human ICOS gene may be used to immunize mice andgenerate antibodies specific for the human T cell inhibitory receptorprotein or ICOS protein, respectively.

Monoclonal antibodies are produced by conventional techniques.Generally, the spleen and/or lymph nodes of an immunized host animalprovide a source of plasma cells. The plasma cells are immortalized byfusion with myeloma cells to produce hybridoma cells. Culturesupernatant from individual hybridomas is screened using standardtechniques to identify those producing antibodies with the desiredspecificity. Suitable animals for production of monoclonal antibodies tothe human protein include mouse, rat, hamster, etc. To raise antibodiesagainst the mouse protein, the animal will generally be a hamster,guinea pig, rabbit, etc. The antibody may be purified from the hybridomacell supernatants or ascites fluid by conventional techniques, e.g.affinity chromatography using the T cell inhibitory receptor bound to aninsoluble support, protein A sepharose, etc.

The antibody may be produced as a single chain, instead of the normalmultimeric structure. Single chain antibodies are described in Jost etal. (1994) J.B.C. 269:26267-73, and others. DNA sequences encoding thevariable region of the heavy chain and the variable region of the lightchain are ligated to a spacer encoding at least about 4 amino acids ofsmall neutral amino acids, including glycine and/or serine. The proteinencoded by this fusion allows assembly of a functional variable regionthat retains the specificity and affinity of the original antibody.

For in vivo use, particularly for injection into humans, it is desirableto decrease the antigenicity of the blocking agent or stimulating agent.An immune response of a recipient against the blocking agent willpotentially decrease the period of time that the therapy is effective.Methods of humanizing antibodies are known in the art. The humanizedantibody may be the product of an animal having transgenic humanimmunoglobulin constant region genes (see for example InternationalPatent Applications WO 90/10077 and WO 90/04036). Alternatively, theantibody of interest may be engineered by recombinant DNA techniques tosubstitute the CH1, CH2, CH3, hinge domains, and/or the framework domainwith the corresponding human sequence (see WO 92/02190).

The use of Ig cDNA for construction of chimeric immunoglobulin genes isknown in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J.Immunol. 139:3521). mRNA is isolated from a hybridoma or other cellproducing the antibody and used to produce cDNA. The cDNA of interestmay be amplified by the polymerase chain reaction using specific primers(U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library ismade and screened to isolate the sequence of interest. The DNA sequenceencoding the variable region of the antibody is then fused to humanconstant region sequences. The sequences of human constant regions genesmay be found in Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, N.I.H. publication no. 91-3242. Human C regiongenes are readily available from known clones. The choice of isotypewill be guided by the desired effector functions, such as complementfixation, or activity in antibody-dependent cellular cytotoxicity.Preferred isotypes are IgG1, IgG3 and IgG4. Either of the human lightchain constant regions, kappa or lambda, may be used. The chimeric,humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab′).sub.2 and Fab may be prepared bycleavage of the intact protein, e.g. by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′).sub.2 fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g. SV-40 earlypromoter, (Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcomavirus LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murineleukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native Igpromoters, etc.

In one embodiment, the blocking agent to a T cell inhibitory receptor isan anti-CTLA-4 antibody that binds to the extracellular domain of CTLA-4and inhibits anti-CTLA-4 signaling. Suitable anti-CTLA-4 antibodies foruse in humans include, e.g., ipilimumab (MDX-010) and tremelimumab (CP675,206). In another embodiment, the blocking agent to a T cellinhibitory receptor is an anti-PD-1 antibody that blocks binding of PD-1to PD-L1 and inhibits PD-1 signaling. Suitable antibodies for use inhumans include, e.g., MDX-1106/ONO-4538 and CT-011. In anotherembodiment, the blocking agent to a T cell inhibitory receptor is ananti-B7-H1 (PD-1L) antibody that blocks binding of PD-1 to PD-1L andinhibits PD-1 signaling. In another embodiment, the blocking agent to aT cell inhibitory agent is a combination of an anti-CTLA-4 antibodyand/or an anti-PD-1 antibody and/or an anti-B7-H1 antibody.

In another embodiment, stimulating agent to ICOS is an anti-ICOSantibody that binds to the extracellular domain of ICOS and activatesICOS signaling, which leads to an increase in T cell activation, e.g.,proliferation. In another embodiment, the stimulating agent to ICOS isrecombinant ICOSL, which may be soluble or expressed on the surface of agenetically modified cell.

Viral Vectors Encoding Blocking or Stimulating Agents and CellsExpressing Same

In one embodiment, the blocking agent(s) to one or more T cellinhibitory receptors and/or the stimulating agent to ICOS is expressedby viral vectors and transformed cells. For example, the viral vectorsand transformed human cells described herein may express anti-T cellinhibitory receptor antibodies to block signaling by the T cellinhibitory receptor and/or a stimulating agent to ICOS (e.g., ICOSligand) that activates ICOS mediated signaling. In a preferredembodiment, the viral vector or human cells expressing the candidateblocking and/or stimulating agent(s) are capable of expressing theagent(s) proximal to a tumor, particularly a tumor infiltratinglymphocyte.

Human cells that can be used include tumor cells, antigen-presentingcells (e.g. dendritic cells), B cells and T cells. The presentlydisclosed cells provide for localized expression of the blocking and/orstimulating agent(s) by cells proximal to a tumor. The cells can bemodified in vivo, or alternatively cells modified ex vivo can beadministered to a patient by a variety of methods, such as by injection.

In one embodiment, the cell is a tumor cell. For ex vivo transformation,such tumor cells can be irradiated to eliminate the ability of the cellto replicate, as known in the art, while maintaining the transientexpression of the blocking and/or stimulating agent(s) afteradministration. For in vivo transformation, non-integrative expressionvectors may be preferred.

In certain preferred embodiments, the tumor cell is autologous orendogenous. In the former instance, the tumor cell is taken from apatient, transfected or transduced with a construct encoding theblocking and/or stimulating agent(s) and re-introduced to the patient,for example after irradiation. In the latter instance, the tumor cell istransformed in vivo by local administration of an appropriate constructas described herein.

In an alternative embodiment, the modified tumor cell is allogeneic. Theallogeneic tumor cell thus can be maintained in a cell line. In thisinstance, the tumor cell can be selected from the cell line, irradiated,and introduced to the patent.

In another alternative embodiment, the modified human cells areantigen-presenting cells such as dendritic cells, or monocytes. Inanother alternative embodiment, the modified human cells are T cells.

Modified human cells capable of producing the blocking and/orstimulating agent(s) can be made by transfecting or transducing thecells with an expression vector encoding the blocking and/or stimulatingagent(s). Expression vectors for the expression of a blocking agent, astimulating agent, or a combination of blocking agent(s) and/orstimulating agents can be made by methods well known in the art.

In various embodiments, the blocking and/or stimulating agent(s) can beadministered to a patient in the form of one or more nucleic acidconstruct.

In one embodiment, the construct comprises a retroviral vector.Retroviral vectors are capable of permanently integrating DNA encodingthe blocking and/or stimulating agent(s) into the cell genome. Thus, inthe case of ex vivo manipulation of autologous or allogeneic cells,stable cell lines that constitutively produce the blocking and/orstimulating agent(s) can be prepared. In a preferred embodiment, thecells are irradiated prior to administration to a patient. Theirradiated cells produce the blocking and/or stimulating agent(s) for alimited period of time

In one embodiment, the expression construct comprises an SFV vector,which demonstrates high levels of transient expression in mammaliancells. The SFV vector is described, for example, in Lundstrom, ExpertOpin. Biol. Ther. 3:771-777 (2003), incorporated herein by reference inits entirety. Thus, in the case of in vivo manipulation of endogenouscells in a patient, transient expression of high levels of the blockingand/or stimulating agent(s) can be accomplished. This is to preventconstitutive expression, and permanent activation, of T cells in vivo.

Systems capable of expressing recombinant protein in vivo are known inthe art. By way of example and not limitation, the system can use the 2Amediated antibody expression system disclosed in Fang et al., NatureBiotech. 23(5) 2005 and U.S. Patent Publication 2005/0003506, thedisclosures of which are expressly incorporated by reference herein intheir entirety. Other systems known in the art are contemplated, and canalso be adapted to produce blocking and/or stimulating agent(s) in vivoas described herein.

Administration of the blocking and/or stimulating agent expressing cellsdisclosed herein can be combined with administration of cytokines thatstimulate antigen-presenting cells such as granulocyte-macrophage colonystimulating factor (GM-CSF)1 macrophage colony stimulating factor(M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3(IL-3), interleukin 12 (IL-12), etc., or cellular vaccines capable ofexpressing such cytokines. In preferred embodiments, the blocking and/orstimulating agent(s) expressing cells are further modified to expresssuch cytokines. Additional proteins and/or cytokines known to enhance Tcell proliferation and secretion, such as IL-1, IL-2, B7, anti-CD3 andanti-CD28 can be employed simultaneously or sequentially with theblocking agents to augment the immune response. The present therapy canalso be combined with any of the molecules, or conducted as describedin, U.S. Pat. No. 6,051,227, incorporated herein by reference in itsentirety.

Vectors and Methods of Transformation

Expression vectors encoding the blocking and/or stimulating agent(s) maybe viral or non-viral. Viral vectors are preferred for use in vivo.Expression vectors of the invention comprise an nucleic acid encoding ablocking agent to a T cell inhibitory receptor or a nucleic acidencoding a stimulating agent to ICOS, or a complement thereof, operablylinked to an expression control region, or complement thereof, that isfunctional in a mammalian cell. The expression control region is capableof driving expression of the operably linked blocking and/or stimulatingagent encoding nucleic acid such that the blocking and/or stimulatingagent is produced in a human cell transformed with the expressionvector.

Expression control regions are regulatory polynucleotides (sometimesreferred to herein as elements), such as promoters and enhancers, thatinfluence expression of an operably linked nucleic acid.

An expression control region of an expression vector of the invention iscapable of expressing operably linked encoding nucleic acid in a humancell. In one embodiment, the cell is a tumor cell. In one embodiment,the cell is a non-tumor cell.

In one embodiment, the expression control region confers regulatableexpression to an operably linked nucleic acid. A signal (sometimesreferred to as a stimulus) can increase or decrease expression of anucleic acid operably linked to such an expression control region. Suchexpression control regions that increase expression in response to asignal are often referred to as inducible. Such expression controlregions that decrease expression in response to a signal are oftenreferred to as repressible. Typically, the amount of increase ordecrease conferred by such elements is proportional to the amount ofsignal present; the greater the amount of signal, the greater theincrease or decrease in expression.

Especially preferred for use in the present invention are induciblepromoters capable of effecting high level of expression transiently inresponse to a cue. When in the proximity of a tumor cell, a celltransformed with an expression vector for the blocking and/orstimulating agent(s) comprising such an expression control sequence isinduced to transiently produce a high level of ICOS ligand by exposingthe transformed cell to an appropriate cue.

Preferred inducible expression control regions include those comprisingan inducible promoter that is stimulated with a cue such as a smallmolecule chemical compound. Particular examples can be found, forexample, in U.S. Pat. Nos. 5,989,910, 5,935,934, 6,015,709, and6,004,941, each of which is incorporated herein by reference in itsentirety.

Expression control regions include full-length promoter sequences, suchas native promoter and enhancer elements, as well as subsequences orpolynucleotide variants which retain all or part of full-length ornon-variant function. As used herein, the term “functional” andgrammatical variants thereof, when used in reference to a nucleic acidsequence, •subsequence or fragment, means that the sequence has one ormore functions of native nucleic acid sequence (e.g., non-variant orunmodified sequence).

As used herein, “operable linkage” refers to a physical juxtaposition ofthe components so described as to permit them to function in theirintended manner. In the example of an expression control element inoperable linkage with a nucleic acid, the relationship is such that thecontrol element modulates expression of the nucleic acid. Typically, anexpression control region that modulates transcription is juxtaposednear the 5′ end of the transcribed nucleic acid (i.e., “upstream”).Expression control regions can also be located at the 31 end of thetranscribed sequence (i.e., “downstream”) or within the transcript(e.g., in an intron). Expression control elements can be located at adistance away from the transcribed sequence (e.g., 100 to 500, 500 to1000, 2000 to 5000, or more nucleotides from the nucleic acid). Aspecific example of an expression control element is a promoter, whichis usually located 5′ of the transcribed sequence. Another example of anexpression control element is an enhancer, which can be located 5′ or 3′of the transcribed sequence, or within the transcribed sequence.

Expression systems functional in human cells are well known in the art,and include viral systems. Generally, a promoter functional in a humancell is any DNA sequence capable of binding mammalian RNA polymerase andinitiating the downstream (3′) transcription of an ICOS ligand codingsequence into mRNA. A promoter will have a transcription initiatingregion, which is usually placed proximal to the 5′ end of the codingsequence, and typically a TATA box located 25-30 base pairs upstream ofthe transcription initiation site. The TATA box is thought to direct RNApolymerase II to begin RNA synthesis at the correct site. A promoterwill also typically contain an upstream promoter element (enhancerelement), typically located within 100 to 200 base pairs upstream of theTATA box. An upstream promoter element determines the rate at whichtranscription is initiated and can act in either orientation. Ofparticular use as promoters are the promoters from mammalian viralgenes, since the viral genes are often highly expressed and have a broadhost range. Examples include the SV40 early promoter, mouse mammarytumor virus LTR promoter, adenovirus major late promoter, herpes simplexvirus promoter, and the CMV promoter.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 31 terminus of the mature mRNA is formedby site-specific post-translattonal cleavage and polyadenylation.Examples of transcription terminator and polyadenylation signals includethose derived from SV40. Introns may also be included in expressionconstructs.

There are a variety of techniques available for introducing nucleicacids into viable cells. Techniques suitable for the transfer of nucleicacid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, polymer-based systems,DEAE-dextran, viral transduction, the calcium phosphate precipitationmethod, etc. For in vivo gene transfer, a number of techniques andreagents may also be used, including liposomes; natural polymer-baseddelivery vehicles, such as chitosan and gelatin; viral vectors are alsopreferred for in vivo transduction (e.g., Dzau et al., Trends inBiotechnology 11, 205-210 [1993]). In some situations it is desirable toprovide a targeting agent, such as an antibody or ligand specific for atumor cell surface membrane protein. Where liposomes are employed,proteins which bind to a cell surface membrane protein associated withendocytosis may be used for targeting and/or to facilitate uptake, e.g.capsid proteins or fragments thereof tropic for a particular cell type,antibodies for proteins which undergo internalization in cycling,proteins that target intracellular localization and enhanceintracellular half-life. The technique of receptor-mediated endocytosisis described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414(1990). For review of gene therapy protocols see Anderson et al.,Science 256, 808-813 (1992).

Where appropriate, gene delivery agents such as, e.g. integrationsequences can also be employed. Numerous integration sequences are knownin the art (see for example Nunes-Duby et al., Nucleic Acids Res.26:391-406, 1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor,Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999;Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). Theseinclude recombinases and transposases. Examples include Cre (Sternbergand Hamilton, J. MoI. Biol., 150:467-486, 1981), lambda (Nash, Nature,247, 543-545, 1974), FIp (Broach, et al, Cell, 29:227-234, 1982) R(Matsuzaki, et al, J. Bacteriology, 172:610-618, 1990), φC31 (see forexample Groth et al., J. MoI. Biol. 335:667-678, 2004), sleeping beauty,transposases of the mariner family (Plasterk et al., supra), andcomponents for integrating viruses such as AAV, retroviruses, andAntiviruses having components that provide for virus integration such asthe LTR sequences of retroviruses or lentivirus and the ITR sequences ofAAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003).

Viral Vectors

In one aspect, the invention provides expression vectors for theexpression of the blocking and/or stimulating agent(s) that are viralvectors. Many viral vectors useful for gene therapy are known (see, forexample, Lundstrom, Trends Biotechnol., 21:117, 122, 2003.

Preferred viral vectors include those selected from the group consistingof Antiviruses (LV), retroviruses (RV), adenoviruses (AV),adeno-associated viruses (AAV), and alpha viruses, though other viralvectors may also be used. For in vivo uses, viral vectors that do notintegrate into the host genome are preferred, such as alpha viruses andadenoviruses, with alpha viruses being especially preferred. Preferredtypes of alpha viruses include Sindbis virus, Venezuelan equineencephalitis (VEE) virus, and Semliki Forest virus (SFV), with SFV beingespecially preferred. See, for example, Lundstrom, Expert Opin. Biol.Then 3:771-777, 2003; Afanasieva et al. Gene Then, 10:1850-59, 2003. Forin vitro uses, viral vectors that integrate into the host genome arepreferred, such as retroviruses, AAV, and Antiviruses.

In a preferred embodiment, the viral vector provides for transient highlevel expression in a transduced human cell.

In one embodiment, the viral vector does not provide for integration ofthe blocking and/or stimulating agent encoding nucleic acid into thegenome of a transduced human cell.

In another embodiment, the viral vector provides for integration of ablocking and/or stimulating agent encoding nucleic acid into the genomeof a transduced human cell.

In one embodiment, the invention provides methods of transducing a humancell in vivo, comprising contacting a solid tumor in vivo with an viralvector of the invention.

In another embodiment, the invention provides methods of transducing ahuman cell ex vivo, comprising contacting a human cell ex vivo with theblocking and/or stimulating agent viral vector of the invention. In oneembodiment, the human cell is a tumor cell. In one embodiment, the humancell is allogeneic. In one embodiment, the tumor cell is derived fromthe patient. In one embodiment, the human cell is a non-tumor cell, suchas, e.g., an antigen presenting cell (APC), or a T cell.

Virus particle coats may be modified to alter specificity and improvecell/tissue targeting, as is well known in the art. Viral vectors mayalso be delivered in other vehicles, for example, liposomes. Liposomesmay also have targeting moieties attached to their surface to improvecell/tissue targeting.

The present application is directed to human cells expressing theblocking and/or stimulating agent. In a preferred embodiment, the humancell expresses a stimulating agent to ICOS (e.g., ICOSL, which may besecreted or expressed as a cell surface protein) that specifically bindsto the extracellular domain of ICOS and activates ICOS mediated negativesignaling. In certain embodiments, the human cell expresses the ICOSligand proximal to a tumor cell for example in a cancer patient. Thus,the human cell is capable of localized expression of the ligand at atumor cell or tumor cell mass. The ICOS ligand can activate ICOSsignaling in cells proximal to said tumor cell, and/or break immunetolerance against a tumor-associated self antigen and stimulate anautoreactive T cell response to said tumor cell. In a preferredembodiment, localized expression of the ICOS ligand reduces or inhibitsundesired adverse immune responses.

It is not necessary for the practice of the invention that the mechanismof action be understood. The cells and methods described herein providehuman cells proximal to tumor cells or tumor cell masses. Expression ofstimulating agents to ICOS and optionally blocking agents to T cellinhibitory proteins or additional cytokines in proximity to the tumorcells enhances anti-tumor immune responses.

Methods of Treatment

Described herein is a method of treating a patient afflicted with acancer comprising administering to the patient a pharmaceuticalcomposition comprising a pharmacologically effective amount of ablocking agent to a T cell inhibitory receptor and stimulating agent toICOS. The method described herein is directed toward the treatment ofcancer, e.g., leukemias and solid tumors (e.g., melanomas, carcinomas,sarcomas, lymphomas, etc.). More common solid cancers include bladdercancer, bone cancer (osteosarcoma), colorectal cancer, brain cancer,breast cancer, cervical cancer, oesophageal cancer, Hodgkin's lymphoma,kidney cancer, liver cancer, lung cancer, mesothelioma, multiplemyeloma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer,penile cancer, prostate cancer, skin cancer (melanoma and non-melanoma)soft tissue carcinoma, gastric cancer, testicular cancer, thyroid cancerand endometrial cancer.

The administered pharmaceutical compositions will often further compriseone or more buffers (e.g., neutral buffered saline or phosphate bufferedsaline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans),mannitol, proteins, polypeptides or amino acids such as glycine,antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylatedhydroxytoluene, butylated hydroxyanisole, etc.), bacteriostats,chelating agents such as EDTA or glutathione, solutes that render theformulation isotonic, hypotonic or weakly hypertonic with the blood of arecipient, suspending agents, thickening agents, preservatives,flavoring agents, sweetening agents, and coloring compounds asappropriate.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the compositions, the type of carrier will typicallyvary depending on the mode of administration. The therapeuticcompositions may be formulated for any appropriate manner ofadministration, including for example, oral, nasal, mucosal, rectal,vaginal, topical, intravenous, intraperitoneal, intradermal,subcutaneous, and intramuscular administration.

For parenteral administration, the compositions can be administered asinjectable dosages of a solution or suspension of the blocking agents ofone or more T cell inhibitory receptors, the stimulating agents of ICOS,an expression vector expressing one or more blocking agents to a T cellinhibitory receptor and/or stimulating agent of ICOS, cells transformedwith expression vectors expressing one or more blocking agents to a Tcell inhibitory receptor and/or stimulating agent of ICOS, or acombination thereof, in a physiologically acceptable diluent with apharmaceutical carrier that can be a sterile liquid such as sterilepyrogen free water, oils, saline, glycerol, polyethylene glycol orethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents, surfactants, pH buffering substances and the likecan be present in compositions. Other components of pharmaceuticalcompositions are those of petroleum, animal, vegetable, or syntheticorigin, for example, non-aqueous solutions of peanut oil, soybean oil,corn oil, cottonseed oil, ethyl oleate, and isopropyl myristate.

The blocking and/or stimulating agents described herein (includingexpression vectors and/or transformed cells expressing such blockingand/or stimulating agents) may be presented in unit-dose or multi-dosecontainers, such as sealed infusion bags, ampoules or vials. Suchcontainers are typically sealed in such a way to preserve the sterilityand stability of the formulation until use. In general, formulations maybe preserved as suspensions, solutions or emulsions in oily or aqueousvehicles, as indicated above. Alternatively, a pharmaceuticalcomposition may be preserved in a freeze-dried condition requiring onlythe addition of a sterile liquid carrier immediately prior to use.

The amount administered to the host will vary depending upon what isbeing administered, the purpose of the administration, such asprophylaxis or therapy, the state of the host, the manner ofadministration, the number of administrations, interval betweenadministrations, and the like. These can be determined empirically bythose skilled in the art and may be adjusted for the extent of thetherapeutic response. Factors to consider in determining an appropriatedose include, but is not limited to, size and weight of the patient, theage and sex of the patient, the severity of the symptom, the stage ofthe disease, method of delivery of the agent, half-life of the agents,and efficacy of the agents. Stage of the disease to consider includeswhether the disease is acute or chronic, relapsing or remitting phase,and the progressiveness of the disease.

Determining the dosages and times of administration for atherapeutically effective amount are well within the skill of theordinary person in the art. For example, an initial effective dose canbe estimated from cell culture or other in vitro assays. A dose can thenbe formulated in animal models to generate a circulating concentrationor tissue concentration, including that of the IC50 as determined by thecell culture assays.

In addition, toxicity and therapeutic efficacy are generally determinedby cell culture assays and/or using experimental animals, typically bydetermining a LD50 (lethal dose to 50% of the test population) and ED₅₀(therapeutically effectiveness in 50% of the test population). Guidanceis found in standard reference works, for example, Goodman & Gilman'sThe Pharmacological Basis of Therapeutics, 10th Ed. (Hardman, J. G. etal., eds.) McGraw-Hill, New York, N.Y. (2001).

For the purposes of this invention, the methods of administration arechosen depending on the condition being treated and the pharmaceuticalcomposition. Administration of the blocking and or stimulating agent(s)can be done in a variety of ways, including, but not limited to,subcutaneously, intravenously, intraperitoneally, intramuscularly, andpossibly direct injection to specified organs or tumors, althoughsystemic administration is preferred. Administration of thepharmaceutical compositions may be through a single route orconcurrently by several routes.

The compositions may be administered once per day, a few or severaltimes per day, or even multiple times per day, depending upon, amongother things, the indication being treated and the judgment of theprescribing physician.

The amount of blocking and/or stimulating agent needed for achieving atherapeutic effect may be determined empirically in accordance withconventional procedures for the particular purpose. Generally, foradministering the cells for therapeutic purposes, the cells are given ata pharmacologically effective dose. “Pharmacologically effective amount”or “pharmacologically effective dose” refers to an amount sufficient toproduce the desired physiological effect or amount capable of achievingthe desired result, particularly for treating the disorder or diseasecondition, including reducing or eliminating one or more symptoms ormanifestations of the disorder or disease. As an illustration,administration of cells to a patient suffering from cancer provides atherapeutic benefit not only when the underlying condition is eradicatedor ameliorated, but also when the patient reports a decrease in theseverity or duration of the symptoms associated with the disease, e.g.,a decrease in tumor burden including disseminated tumor cells (DTC), adecrease in circulating tumor cells, an increase in progression freesurvival. Therapeutic benefit also includes halting or slowing theprogression of the underlying disease or disorder, regardless of whetherimprovement is realized. Pharmacologically effective dose, as definedabove, will also apply to therapeutic compounds used in combination withthe cells, as further described below.

Preferably, the effect will result in a quantifiable change of at leastabout 10%, preferably at least 20%, 30%, 50%, 70%, or even 90% or more.Therapeutic benefit also includes halting or slowing the progression ofthe underlying disease or disorder, regardless of whether improvement isrealized. When the combination of a blocking agent of a T cellinhibitory receptor and a stimulating agent of ICOS is used in withother treatment protocols, an effective amount is in ratio to acombination of components and the effect is not limited to individualcomponents alone.

A pharmacologically effective amount that will treat cancer willmodulate the symptoms typically by at least about 10%; usually by atleast about 20%; preferably at least about 30%; or more preferably atleast about 50%. Such will result in, e.g., statistically significantand quantifiable changes in the numbers of cells being affected. Thismay be a decrease in the numbers of micrometastases in distant organs, adecrease in recurrent metastatic disease, etc.

The blocking and stimulating agents described herein may be combinedwith other antitumor treatments, e.g., surgical resection, radiationtherapy, chemotherapy, immunotherapy, and supportive therapy (e.g.,painkillers, diuretics, antidiuretics, antivirals, antibiotics,nutritional supplements, anemia therapeutics, blood clottingtherapeutics, bone therapeutics, and psychiatric and psychologicaltherapeutics). Such other antitumor treatments, including treatment withone or more blocking agents to one or more T cell inhibitory receptors,may be provided sequentially (e.g., before or after) or simultaneouslywith the administration of the stimulating agent of ICOS.

EXAMPLES Example 1: Stimulating Agents to ICOS Enhance the Anti-TumorEffects of Anti-CTLA-4 Antibody and Anti-PD-L1 Antibody Example 1.1:Effect of a Stimulating Agent to ICOS Antibody on CD4⁺ T CellProliferation

CD4+ T cells were prepared from C57BL/6 mice spleen by Dynal murine CD4+T cell negative selection kit according to the manufacturer'sinstruction. Fifty thousand CD4+ T cells were stimulated in a 96 wellplate pre-coated with or without anti-CD3 mAb (0.5 μg/ml) and 2 μg/ml ofanti-CD28, 5 μg/ml of anti-ICOS mAb (clones C398.4A and 7E.17G9). Cellswere incubated at 37° C., in a 5% of CO₂, for 72 hr and 1 μci of3H-thymidine was added into each well 8 hr before the end of theculture. Plate was harvested and analyzed for 3H-thymidineincorporation.

As shown in FIG. 1, anti-ICOS antibodies enhanced proliferation of CD4⁺T cells in the presence of anti-CD3 antibody.

Example 1.2: Indirect Correlation Between Anti-CTLA-4 Induced ICOSExpression and Tumor Growth

Mice were challenged with 2×10⁴B16/F10 tumor cells. Mice were untreatedor treated. Treated animals received 200 μg anti-CTLA-4 antibody on day3 and 100 μg anti-CTLA-4 antibody on days 6, 9, 12, 15, 18, and 21 posttumor challenge. Tumor growth and levels of ICOS on CD4⁺FOXP3⁻ effectorT cells in the blood were monitored every three days.

As shown in FIG. 2, CD4⁺FOXP3⁻ cells isolated from treated animalsexpressed increased levels of ICOS. Additionally, the increasedexpression of ICOS indirectly correlated with tumor burden (FIG. 2).

Example 1.3: ICOS⁻ or ICOSL⁻ Mice Demonstrated a Reduced Anti-TumorResponse Mediated by Anti-CTLA-4 Antibody

Wild type C57BL/6, ICOS deficient C57BL/6, and ICOS-ligand (ICOSL)deficient C57BL/6 mice bearing B16/BL6 tumors were either left untreatedor treated s.c. (on day 3 post-tumor implantation) with 1×10⁶ irradiatedGM-CSF-producing B16 (GVAX) and anti-CTLA-4 i.p. (9H10), at a dosing of0.2, 0.1 and 0.1 mg on days 3, 5 and 7, respectively. Tumor growth wasmonitored and percent survival calculated on day 80.

Wild type, ICOS deficient, or ICOSL deficient mice bearing tumors andleft untreated died between 25 and 41 days after tumor implantation(open circle, open triangle and open square respectively). Conversely,90% survival was observed when wild type mice were treated with GVAX andanti-CTLA-4 combination therapy (closed circles). Remarkably, ICOSdeficient (closed triangles) and ICOSL deficient mice (closed squares)showed significantly lower protection after being treated with GVAX andanti-CTLA-4 antibody, demonstrating a key role for this ligand/receptorpair interaction during GVAX/anti-CTLA-4 combination therapy.

Example 1.4: Enhanced Anti-Tumor Effect Using GVAX, Anti-CTLA-4Antibody, and Anti-ICOS Antibody

Mice challenged with 5×10⁴B16/BL6 tumor cells were (1) untreated, (2)treated with 1×10⁶ irradiated GM-CSF-producing B16 only (GVAX; s.c. 3,6, and 9 days post-implantation), (2) treated with 1×10⁶ irradiated GVAX(s.c. 3, 6, and 9 days post-implantation), 200 μg anti-CTLA-4 antibody(day 3 post-tumor implantation), and 100 μg anti-CTLA-4 antibody (days6, 9, 13, and 17 post-tumor implantation), or (3) treated with 1×10⁶irradiated GVAX (3, 6, and 9 days post-implantation), 200 μg anti-CTLA-4antibody (day 3 post-tumor implantation), 100 μg anti-CTLA-4 antibody(days 6, 9, 13, and 17 post-tumor implantation) and 200 μg anti-ICOSantibody (days 3, 6, 9, 13, and 17 post-tumor implantation). Tumorgrowth was monitored and survival was calculated on day 80.

As shown in FIG. 4, treating animals with a combination of GVAX,anti-CTLA-4 antibody and anti-ICOS antibody resulted in delayed tumorgrowth compared to treating animals with GVAX and anti-CTLA-4 antibodyonly. This finding is consistent with the finding that mice treated withGVAX with anti-CTLA-4 and anti-ICOS antibodies exhibit higher survivalrates compared to mice treated with GVAX and anti-CTLA4 antibody only(FIG. 5).

Example 1.5: Enhanced Anti-Tumor Effect Using Anti-ICOS and Anti-PD-L1Antibodies

Three-day B16/BL6-bearing mice were either untreated or treated s.c. (onday 3 post-tumor implantation) with 1×10⁶ irradiated GM-CSF-producingB16 (GVAX) and i.p. anti-ICOS antibody (7E.17G9), anti-PD-L1 antibody(10F.9G2) or the combination at a dosing of 0.2, 0.1 and 0.1 mg on days3, 5 and 7 respectively. Tumor growth was monitored and percent survivalcalculated on day 80.

Mice treated with a combination of GVAX and anti-ICOS antibody, or GVAXand anti-PD-L1 antibody demonstrated poor survival rates (FIG. 6). Incontrast, combination therapy using anti-PD-L1 antibody, anti-ICOSantibody and GVAX resulted in 50% survival, demonstrating a potentsynergistic effect obtained with the combination of anti-ICOS antibody(7E.17G9) with anti-PD-L1 antibody (10F.9G2) and GVAX.

Example 2: Use of ICOS Ligand Expressing Tumor Cells as an Anti-TumorVaccine Example 2.1 Example 2.1.1: Antibodies

Anti-CTLA4 (clone 9H10) was purchased from Bio X Cell.

Example 2.1.2: Cell Lines

The highly tumorigenic and poorly immunogenic melanoma cell line B16/BL6was used for tumor challenge. B16/BL6-expressing GM-CSF, here referredto as GVAX, was used for treatment of tumor-bearing mice. B16-Thy1.1 wasgenerated through retroviral transduction of B16/BL6 cells with thevector MSCV-IRES-Thy1.1 which was a gift from Dr. Leo lefrancois atUniversity of Connecticut. B16-mICOSL was generated through retroviraltransduction of B16/BL6 cells with the vector MSCV-ICOSL expressing fulllength of mouse ICOSL (gift from Dr. William Sha, University ofCalifornia, Berkeley). GVAX cells were also transduced with theMSCV-ICOSL vector to generate GVAX-mICOSL.

Example 2.1.3: Tumor Challenge and Treatment Experiments

Mice were injected in the right flank i.d. on day 0 with 50,000 B16/BL6melanoma cells and treated on days 3, 6, 9, and 12 with 7.5×10⁵irradiated (150 Gy) GVAX mixed with 7.5×10⁵ irradiated (150 Gy)B16/BL6-Thy1.1 (n=10) or B16-mICOSL (n=10) on the left flank, incombination with 100 μg anti-CTLA4 i.p. (200 μg on day 3). Tumor growthand rejection were monitored over time.

Mice were injected in the right flank i.d. on day 0 with 20,000 B16/BL6melanoma cells and treated or not on days 3, 6, 9, and 12 with 1×10⁶irradiated (150 Gy) GVAX (n=10) or GVAX-mICOSL (n=10) on the left flank,in combination with 100 μg anti-CTLA4 i.p. (200 μg on day 3). Tumorgrowth and rejection were monitored over time.

Mice were injected in the right flank i.d. on day 0 with 20,000 B16/BL6melanoma cells and treated or not on days 3, 6, 9, and 12 with 1×10⁶irradiated (150 Gy) B16/BL6-Thy1.1 (n=10) or B16-mICOSL (n=10) on theleft flank, with or without 100 μg anti-CTLA4 i.p. (200 μg on day 3).Tumor growth and rejection were monitored over time.

Mice were injected in the right flank i.d. on day 0 with 20,000 B16/F10melanoma cells and treated or not on days 3, 6, 9, and 12 with 1×10⁶irradiated (150 Gy) B16-mICOSL (n=5) on the left flank, with or without100 μg anti-CTLA4 i.p. (200 μg on day 3). Tumor growth and rejectionwere monitored over time.

Example 2.2: Results

The B16 cellular vaccine expressing ICOSL, in the absence or presence ofGVAX, did not add to the tumor protection rate beyond the previouscombination therapy of GVAX and CTLA-4 blockade (FIGS. 7 and 8).

In the setting without GM-CSF, the B16 cellular vaccine expressing ICOSLhas a synergistic effect together with CTLA-4 blockade to provide delayin tumor growth and/or overall advantage in tumor rejection (FIGS.9-12).

All patents and patent publications referred to herein are herebyincorporated by reference.

Certain modifications and improvements will occur to those skilled inthe art upon a reading of the foregoing description. It should beunderstood that all such modifications and improvements have beendeleted herein for the sake of conciseness and readability but areproperly within the scope of the following claims.

What is claimed is:
 1. A method of treating cancer in a patientcomprising administering to the patient a blocking anti-CTLA-4 antibodyand an agonist anti-ICOS antibody.
 2. The method of claim 1, wherein theblocking anti-CTLA-4 antibody and the agonist anti-ICOS antibody areadministered simultaneously or sequentially.
 3. The method of claim 1,wherein the agonist anti-ICOS antibody is a monoclonal antibody.
 4. Themethod of claim 3, wherein the blocking anti-CTLA-4 antibody is amonoclonal antibody.
 5. The method of claim 4, wherein the agonistanti-ICOS antibody is a humanized antibody.
 6. The method of claim 3,wherein the blocking anti-CTLA-4 antibody is a humanized antibody. 7.The method of claim 1, wherein the agonist anti-ICOS antibody is ahumanized antibody.
 8. The method of claim 7, wherein the blockinganti-CTLA-4 antibody is a humanized antibody.
 9. The method of claim 1,consisting of administering to the patient one or more doses of theblocking anti-CTLA-4 antibody and one or more doses of the agonistanti-ICOS antibody.
 10. The method of claim 9, wherein at least one doseof the blocking anti-CTLA-4 antibody and at least one dose of theagonist anti-ICOS antibody are administered simultaneously orsequentially.
 11. The method of claim 9, wherein the agonist anti-ICOSantibody is a monoclonal antibody.
 12. The method of claim 11, whereinthe blocking anti-CTLA-4 antibody is a monoclonal antibody.
 13. Themethod of claim 12, wherein the agonist anti-ICOS antibody is ahumanized antibody.
 14. The method of claim 11, wherein the blockinganti-CTLA-4 antibody is a humanized antibody.
 15. The method of claim 9,wherein the agonist anti-ICOS antibody is a humanized antibody.
 16. Themethod of claim 15, wherein the blocking anti-CTLA-4 antibody is ahumanized antibody.
 17. The method of claim 4, wherein the method doesnot comprise administering another antitumor treatment.
 18. The methodof claim 4, wherein the method does not comprise administering anotherantitumor treatment other than surgical resection or radiation therapy.19. The method of claim 4, wherein the method does not compriseadministering another immunotherapy.